In general, a biosensor is a device capable of identifying a target biomolecule such as a polynucleotide, polypeptide, or other biomolecule of interest. There is great interest in developing biosensors to be used for varied purposes from monitoring gene expression in organisms to identification and speciation of possible pathogens and/or biocontaminants to the identification of drug candidates. Such devices would be a great benefit for food and water safety monitoring, medical diagnostics, and defense of military and civilian populations from biological threats. Sensors developed for the detection of biological analytes are typically based on ligand specific binding events between a recognition binding pair, such as antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, and complementary nucleic acid strands. The analyte to be detected may be either member of the binding pair, or the target analyte may be a ligand analog that competes with the ligand for binding to the complement receptor.
Traditional biosensors designed for the purpose of detecting binding events between complementary binding pairs, such as those described above, are large, and require significant volumes of liquid reagents and highly trained personnel. Typically, the reduction or elimination of any of these requirements leads to a subsequent loss of sensitivity and/or selectivity. Over the past several years researchers have been striving to develop alternatives to current biosensor technologies, but many developments have been geared to large, array-based equipment to increase sensitivity or throughput in the laboratory setting.
Examples of some of these efforts include optical biosensors that employ recognition elements to detect a target analyte, such as nucleic acid (e.g., DNA or RNA) hybridization assays. Such hybridization assays have been developed to interrogate samples for multiple analytes from a single sample. Nucleic acid based biosensors can be very selective; however, the optical techniques employed in many such sensors require multiple liquid reagents which must be stored in controlled environments and fluorescent labels that can be unstable. Labeling of biological molecules can be very expensive and produce low yields. Also the need for optics to excite or collect fluorescent signal adds expensive and complicated components and creates alignment issues. It would be desirable to reduce the reagent load, remove the fluorescent labels, reduce manufacturing and operational costs and make the sensing element reusable.
Biosensors incorporating electrochemical techniques were developed in order to meet some of these needs. A typical electrochemical biosensor includes a base electrode and a biochemically discriminating element in contact or otherwise coupled to the electrode. The biochemically discriminating element functions either to detect and transform the target analyte into an electrically active species, which is then detected by the electrode, or to otherwise generate an electrical signal, which is sensed and monitored by the electrode.
The application of electrochemical techniques to biosensor technology holds many advantages over optical techniques including, but not limited to, the lack of optical elements to align, the ability to operate in turbid media such as blood or waste water, as well as the ability to capitalize on the vast electronics processing industry for electrode arrays and control electronics. However, manufacture and use of such electrochemical biosensors has proven challenging due to complicated designs and electrochemical interferences caused by interactions of substances other than the target analyte.
Due to the difficulty of converting a biochemical binding event into an electrochemical signal, early applications of electrochemical biosensors were designed for detecting analytes that are themselves electrochemical species, or that can participate in reactions that generate electrochemical species, rather than to direct detection of ligand-receptor binding events. However, such sensors are quite limited in their application. In an effort to overcome this problem, sensors were developed that involve an intermediate reaction or a secondary active species of some sort, which acts to generate the electrochemical signal. One such design includes two separate reaction elements in the biosensor: a first element contains a receptor and bound enzyme-linked ligand, and the second element includes components for enzymatically generating and then measuring an electrochemical species. In operation, analyte ligand displaces the ligand-enzyme conjugate from the first element, releasing the enzyme into the second element region, thus generating an electrochemical species which is measured in the second element. Two-element biosensors of this type are relatively complicated to produce, thus limiting their usefulness.
Biosensors that attempt to couple electrochemical activity directly to a ligand-receptor binding event without the use of two reaction elements have been proposed where a lipid bi-layer membrane containing an ion-channel receptor that is either opened or closed by ligand binding to the receptor controls access to the electrode. Electrodes of this type are also complicated, difficult to make and store, and are limited at present to a rather small group of receptor proteins.
As discussed, many of the above techniques have disadvantages such as complicated design, expensive reagents and manufacturing costs, use of fluorescent tags, applicability to only a small class of biomolecules, and complicated, multi-step processing. Thus, there is a need in the industry for a biosensor that overcomes at least these disadvantages, such as a biosensor utilizing porous materials attached to electrodes.
Examples of biosensor applications include U.S. Pat. Nos. 5,589,396 (Frye, et. al.), 5,624,537 (Turner, et. al.), 6,332,363 (Molloy, et. al.), 6,485,987 (Charych, et al.), 6,551,496 (Moles, et. al.), 6,800,448 (Rider et. al.), 6,960,466 (Pamidi, et. al.), and examples of porous materials include U.S. Pat. Nos. 4,286,087 (Austin, et. al.), 4,895,724 (Cardinal, et. al.), 5,015,293 (Mayer, et. al.), 5,525,710 (Unger, et. al.), 5,620,706 (Dumitriu, et. al.), 5,830,883 (Block, et. al.), 5,840,341 (Watts, et. al.), 5,871,985 (Aebischer, et. al.), and 6,773,723 (Spiro et. al.), the teachings all of the above U.S. Patents are fully incorporated by reference.